BIOCHEMISTRY

Plenary: Column and Paper LE1

Chromatography
Dra. MACarlos || 08/23/2017

OUTLINE B. TYPES OF CHROMATOGRAPHY

I. Introduction
A. Chromatography COLUMN CHROMATOGRAPHY
i. Stationary Phase
ii. Mobile Phase ● Common method of separating and isolating mixtures of
B. Types of Chromatography proteins and nucleic acids
i. Ion Exchange Chromatography ● Differs in the type and components of the two phases
a. Cation Exchangers
b. Anion Exchangers Ion Exchange Chromatography
ii. Paper Chromatography ● Uses the net charge of each component as a basis for
C. Retention Factor separation and extraction
D. Isoelectric Point ● Opposite charges attract, same charges repel
II. Experiment results ● The amino acid that has a net charge opposite to the resin
III. Guide Questions would be attracted to the resin while the amino acid with a net
charge that is opposite to the buffer would be attracted to the
buffer and will move along with it.
LEARNING OBJECTIVES
● Separates molecules based on size, charge and polarity
I. Explain the principle involved in the separation of an unknown
● The following influences the separation capacity of the setup
mixture of amino acids by column and paper chromatography;
→ Net surface charge of a protein at a certain pH
II. Differentiate cationic exchangers from anionic exchangers
▪ As the pH of the buffer approaches the pI of the molecule,
III. Describe the effect of different pH of buffers on the electrical
the sample components confer a neutral net charge thus
charges of amino acids;
lowering its affinity to the stationary phase (this allows
IV. Discuss how pKa of amino acids relate in the changes of buffer
efficient elution of the sample components).
pH in separating the components of amino acid mixtures;
→ pH of the buffer
V. Explain the principle in paper partition chromatography; and
▪ Use a buffer with a pH 0.05-1.00 pH units from the pI
VI. Discuss the importance of Retention factor.
of the protein
INTRODUCTION → Ionic strength of the buffer
→ Elution conditions
A. CHROMATOGRAPHY ● Components
● Physicochemical method for separating complex mixtures by → Stationary Phase: Ion exchange resins
distributing the components between two phases → Mobile Phase: Buffers (varying pH)
● Separation of molecules is based on the interaction of the target ● Types of IEC
molecules with the different components of the chromatography → Cationic Exchangers
● Phases ▪ The resin components are negatively charged that allows
→ Stationary phase positively charged (cations) substances to attach
▪ A solid or liquid substance that is fixed and acts as a ▪ Utilizes Weakly acidic groups
constraint to the motion of the components of a mixture → Anionic Exchangers
→ Mobile phase (Eluent) ▪ The resin components are positively charged that allows
▪ Liquid or a gas that passes through the stationary phase negatively charged (anions) substances to attach.
that carries the target molecules based on the ▪ Utilizes weakly basic groups
interaction between the two substances
● Eluate
→ Liquid solution that results from the chromatography setup

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 Cationic ▪ Factors affecting capillary action:
Parameter Anionic Exchanger
Exchanger − Cohesion: attractive forces between similar molecules
Acidic Group Basic Group − Adhesion: attractive forces between dissimilar
Functional Groups
(Sulfuric/COOH) (NH2/NH4) molecules
Charge of Positive Charge − Surface Tension: tension of the surface film of liquid
Negative Charge
functional group due to attraction of particles in the surface layer
Increasing pH of Sequence of ph Decreasing pH of o ↑SA = A > C
Buffer Buffer Buffer
AA Sequence AA Sequence
Sequence of AA
(Acidic, Neutral, (Basic, Neutral,
Eluted
Basic) Acidic)

Table 1. Comparison of anionic and cationic exchangers lifted from

the lecture of Dra. MACarlos.
● If the sample components are more stable below their pI, use a
cation exchange medium
● If the sample components are more stable above their pI, use
an anion exchange medium

Figure 2 Ascending Paper Chromatography

● Descending Paper Chromatography
→ Solvent travels downwards on the paper through GRAVITY
→ Faster due to the aid of gravity

Figure 1. Diagram of the elution process in Ion Exchange Chromatography

PAPER CHROMATOGRAPHY
● Mechanism of Action
→ Separates components of a mixture, based on differences in
affinities (polarity) to water and mobile phase (solvent front)
during movement.
→ Compounds get separated because of their relative polarity
and weight.
● Components
→ Stationary Phase: Paper (cellulose)
→ Mobile Phase: Solvent (alcohol) Figure 3 Descending Paper Chromatography

● Principles:
RETENTION FACTOR
→ MORE SOLUBLE the component = DISSOLVES FASTER in
● Ratio of the distance traveled by the substance to the distance
the solvent and carried along by the solvent
traveled by the solvent.
→ The MOST POLAR molecule travels the SHORTEST
DISTANCE because the paper is also polar (cellulose = → Rf = 0 Substance dissolves to the stationary phase, and thus
carbohydrate; contains a lot of OH groups). Therefore, they stays on the starting point.
interact with each other strongly (primarily because of → Rf = 1 Substance has no affinity to stationary phase, and
hydrogen bonding). thus travels with the mobile phase.
→ The MOST NON-POLAR molecule travels the FARTHEST. ● Measuring Distance:
→ In the case of molecules with very close polarity, weight will → Distance moved by the solute: From the spotting line to the
determine which molecule travels further (lighter weight = spot
greater distance traveled). → Distance moved by the solvent: From the spotting line to the
solvent front line.
● 2 Types of Paper Chromatography:
→ Ascending Paper Chromatography
▪ Solvent travels upward on the paper through CAPILLARY
ACTION
▪ Slower
▪ Velocity of rise declines with time

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 ● Increase in basicity and acidity (change in pH) would alter the
concentrations of [HA] and [A-]
→ Creating a stronger base and stronger acid respectively.
● If the pH is greater than the pI, the amino acid confers a
negative charge.
● If the pH is less than the pI, the amino acid is positively charged

EXPERIMENTAL RESULTS
● Retention Factor and Identification of unknown
→ AMINO ACIDS:
Figure 4 Example of computing the Rf value ▪ Standard 1: Alanine (Rf = 0.57)
● Example: ▪ Standard 2: Aspartic acid (Rf = 0.42)
● Solvent (77% ethanol): Rf = 195 cm ▪ Standard 3: Arginine (Rf = 0.38)
→ Std 1 (Ala): Rf = 112/195 = 0.57
→ Std 2 (Asp): Rf = 81/195 = 0.42 ● IONIZATION OF AMINO ACIDS:
→ Std. 3 (Arg): Rf = 75/195 = 0.38
● Trend: Decreasing

Amino Acid Covalent Bond Molecular Molecular

Weight (g) Size
Alanine (Ala) Non-polar 89.09 Smallest Figure 4. Alanine
Aspartic Acid Polar 133.10 Smaller than
(Asp) Arginine
Arginine Polar; Slightly 174.20 Largest
(Arg) soluble in Ethanol
Table 2. Comparison of anionic and cationic exchangers lifted from the
lecture of Dra. MACarlos.
● Factors Affecting Rf Value:
→ Temperature Figure 5. Aspartic acid
→ Purity & Type of solvent/s used
→ Quality of the paper, adsorbents & impurities present in the
adsorbents
→ Chamber saturation techniques, method of drying &
development
→ Distance travelled by the solute & solvent
→ Chemical reaction between the substances being partitioned
→ Size/molecular weight of molecule

● Sources of Error
→ Error during application of the spots Figure 6. Arginine
→ minimum volume of the concentrated solution to avoid STUDY GUIDE QUESTIONS
diffusion through the paper which leads to poor separation
→ Spots should be approximately of the same diameter. 1. Summarize the principle involved in the separation of
● Development
amino acids by ion exchange chromatography.
→ Improper placement of the paper in the tank
a. Differentiate cationic exchange column from anionic
→ Chamber saturation (5 – 7 hours)
● Detection exchange column.
→ Spraying methods affect the final result (Ninhydrin test)
→ Time of measurement of Rf Answer:
In cationic exchange column, it utilizes resins that are
negatively charged (weakly acidic groups) to attract cationic
ISOELECTRIC POINT
substances whereas in anionic exchange column, it utilizes resins
● pH at which the protein has an overall zero net charge (Zwitter
that are positively charged (weakly basic groups) to attract anionic
ion form)
substances.
● average pKa of each amino acid
→ pI=(pK1+ pK2)/2
b. Explain briefly and identify the two phases present in the
● pKa is the pH of the buffer (concentration of the two buffering
chromatography.
species are equal and max buffering capacity)
→ pKa = pH + log [HA] / [A-]
→ pH = pKa
 then log [HA] / [A-] = 0
→ [HA] = [A-]

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Answer: Ascending Descending
Mobile phase: Liquid solvent which contains substances to be Solvent Flow Upward Downward
identified that traverses over the stationary phase. Flow Rate Slower Faster
• Buffer Solutions (Ion-exchange chromatography) Driving Force Capillary Action Capillary Action +
• Solvents (Paper Partition Chromatography) Gravity
Stationary phase: non-moving phase. Setup Solvent rises from Solvent travels from
the bottom of the the top of the
• Resin (Ion-exchange chromatography)
chamber to the top chamber to the
• Cellulose paper (Paper Partition Chromatography) bottom
2. Summarize the principle involved in the identification of
6. Enumerate factors affecting the distance travelled by
unknown amino acids by paper chromatography.
an amino acid in a chromatogram.
Answer:
● Size of the solute
Amino acids travel at varying distances in paper chromatography → Amino acids with smaller side chains travel faster than those
due to its structural properties: polarity, solubility and molecular with larger side chains
weight. Unkonwn amino acids can be identified by matching and ● Polarity
comparing the computed Rf values of unknown amino acids to the → Amino acids which have higher affinity and similar polarity for
standard Rf values of amino acids. the solvent will have higher solubility in the solvent and travel
faster and farther. Thus, Non-polar amino acids will have
3. Three amino acids in a solution are to be fractioned by ion- higher Rf values in non-polar solvent.
exchange chromatography using Dowex 50-X4. ● pH
Amino acid pI → The pH of the solvent will determine the charge of the amino
Glycine 5.97 acid. More charged amino acids have higher affinity to polar
Histidine 7.59 solvents while less charged amino acids have lower affinity.
Glutamate 3.22 ● Temperature
The amino acid mixture is placed in the column where three → Solubility of the amino acid in the solvent increases with
buffers were passed successively. The pH values of the temperature thus traveling farther and leading to higher Rf
eluting buffers are as follows: Values
Eluting Buffer pI REVIEW QUESTIONS
1 3.4 1. Which of the following statements are not true:
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a.Stationary phase in a chromatography is a liquid that
3 9
a. Which amino acids were attached to the column initially? constrains the motion of the components of the
Explain. mixture.
Initially, all amino acids were attached to the column. b. IEC separates molecules based on their size, charge
Amino acids are protonated in acidic environment thus attracted to and polarity.
the Dowex 50-X4 (cationic column) which is negatively charged. c. An Anionic Exchanger contains acidic functional
groups, hence giving it a negative charge.
b. State which amino acids were eluted by each successive d.Cationic Exchangers elute amino acids with high [H+]
buffer. to amino acids with low [H+].
The addition of buffer solutions makes amino acids to be 2. In what condition will the amino acid be eluted out
eluted upon reaching their isoelectric point (pH beyond pI). Buffer easier:
1 elutes Glutamate; buffer 2 elutes Glycine; buffer 3 elutes a. The net charge of the amino acid is (+) in a highly (-)
Histidine.
stationary phase.
b. The pH of the buffer used is 2.5 pH units below the pI
4. In the paper chromatography experiment, why was it
of the target molecule/ amino acid.
necessary to use standard amino acids to identify
unknown amino acids? c. A cationic exchanger is used to elute a highly basic
Numerous factors such as properties of the stationary amino acid.
phase and the mobile phase affect the migration of eluents in a d.A buffer with (+) charge is used to elute an amino
paper chromatography column which may result in difference of acid with a neutral net charge.
the actual retention factors of the eluates from their theoretical Answers: c, d,
retention factors. Thus, amino acids with previously known REFERENCES
retention factors known as standards must be used to be able to Biochemistry Laboratory Manual. Department of
identify unknown amino acids by comparing their retention factors. Biochemistry, UERMMCI
5. Differentiate between ascending and descending Class Powerpoint
paper chromatography. 2020 Transcriptions

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